Infertility afflicts approximately 10-15% of couples and has causes linked to all the major events in the reproductive pathways, from development of functional eggs and sperm, to fertilization, embryonic transcriptional activation, implantation, placentation and growth of a viable fetus. In spite of the frequency of infertility and impact on the quality of life of those affected, little is known of underlying molecular and genetic causes. Nonethless, recent advances in assisted reproductive techniques, reproductive biology, human embryonic stem cell (hESC) biology and human genetics, may now allow us to overcome two historicallysignificant limitations in human reproductive studies: namely, the inaccessibility of early human development to biological exploration and the genetic-intractability of the human genome during development. In this proposal, we use the powerful tools of hESC biology and human genetics to test the hypotheses: 1) that multiple members of the DAZ (Deleted in AZoospermia) gene family are required for the formation and/or maintenance of nascent human germ cells and 2) that common genetic variants and unique mutations, enriched in chromosomes of infertile men and women, modulate early human germ cell development in both sexes. Our specific aims are: Aim 1. Define the baseline molecular, temporal and genetic characteristics of differentiation of hESCs and mESCs to the germ cell lineage in vitro and in.vivo. Aim 2. Silence human DAZ, and human and mouse DAZL and BOL genes and assess germ cell development. Aim 3. Examine functional significance of a common variant and a rare mutation in the DAZ gene family in the human population and relate results to human genetic studies and clinical findings. This research is particularly responsive to the health concerns of infertile couples who seek assisted reproductive technologies in hopes of achieving biological parenthood. Indeed, successful completion of the proposed research promises to strengthen our basic understanding of the remarkable pathways by which human germ cells develop, provide useful tools for basic scientists to study human germ cell development, and contribute to the development of novel therapeutics and validated diagnostic genetic tests for clinical use. The research would greatly benefit from the establishment of a center that provides interactions across the community and access to valuable core facilities. This research uses hESC lines from the NIH Registry (UC01, UC06, WA01 and WA09).